Sodium ion-driven chloride /bi-carbonate exchanger

ABSTRACT

The present invention discloses DNAs comprising nucleotide sequences set forth as SEQ ID NO: 1 or NO: 3 encoding Na + -driven Cl—/HCO 3 — exchanger, proteins comprising amino acid sequences set forth as SEQ ID NO: 2 or NO: 4, and their homologous proteins comprising an amino acid sequence having deletion, substitution, addition or insertion amino acids, which proteins, when expressed in a cell, functions as Na + -driven Cl—/HCO 3 — exchanger, and cells in which the proteins exogenously expressed.

FIELD OF THE INVENTION

[0001] The present invention relates to human and mouse Na⁺-drivenCl—/HCO₃— exchanger (sodium ion-driven chloride/bicarbonate exchanger)proteins, which are a class of proteins involved in intracellular pHregulation. More specifically, the present invention relates to sodiumion-driven chloride/bicarbonate exchanger proteins, cells designed toexpress one of the proteins, which cells are of a species different fromthe origin of the one of the proteins expressed, DNAs encoding theproteins, antibodies to the proteins, and a method for selectingagonists/antagonists of the sodium ion-driven chloride/bicarbonateexchanger proteins.

BACKGROUND OF THE INVENTION

[0002] Regulation of intracellular pH (pH_(i)) in response to variousstimuli is a critical one among a number of cellular functions. A familyof bicarbonate transporters is a major pH_(i) regulator underphysiological conditions in animal cells. Bicarbonate (HCO₃—)transporters are divided into four groups according to their functions[Boron, W. F. et al., J. Exp. Biol., 200:263-268(1997)]: Na⁺-independentCl—/HCO₃— exchanger (alternatively called an anion exchanger, AE),Na⁺—HCO₃— cotransporter (NBC), K⁺—HCO₃— cotransporter, and Na⁺-drivenCl—/HCO₃— exchanger. Three AEs and three NBCs have been cloned andfunctionally characterized, but the molecular structure of the K⁺—HCO₃—cotransporter and the Na⁺-driven Cl—/HCO₃— exchanger have remainedunknown.

[0003] A Na⁺-driven Cl—/HCO₃— exchanger was first discovered ininvertebrate neurons and was later found in vertebrate neurons as wellas non-neuronal cells, including brain, vascular endothelial cells,sperm, kidney and pancreatic β-cells. Na⁺-driven Cl—/HCO₃— exchanger isan intracellular pH regulator that transports extracellular Na⁺and HCO₃—into the cells in exchange for intracellular Cl—, thereby playing animportant role in cellular alkalinization.

[0004] In pancreatic β-cells, glucose is the most importantphysiological regulator of insulin secretion. Glucose metabolism inducesan increase in intracellular pH in the pancreatic cells. It has beenshown that this glucose-induced pH_(i) rise is evoked primarily by theaction of Na⁺-driven Cl—/HCO₃— exchanger [Pace, C. S. et al., J.Membrane Biol., 73:39-43(1983)].

[0005] Na⁺-driven Cl—/HCO₃— exchanger is thus an important intracellularpH regulator in various cells, but its molecular basis is not known.Analysis of the molecular structure and function of Na⁺-driven Cl—/HCO₃—exchanger should be valuable not only for functional analysis of insulinsecretion by pancreatic β-cells but also for screening as well as fordrug designing based on its molecular structure aimed at the developmentof therapeutics of diabetes mellitus.

[0006] On the above background, the present invention has as itsobjective to clone Na⁺-driven Cl—/HCO₃— exchangers, thereby obtainingtheir DNA for sequencing, providing cells of a different speciesexpressing the DNAs, and determining the structure and function of theNa⁺-driven Cl—/HCO₃— exchangers.

SUMMARY OF THE INVENTION

[0007] Thus, the present invention provides a Na⁺-driven Cl—/HCO₃—exchanger protein comprising the amino acid sequence set forth as SEQ IDNO: 2 or NO: 4 in the Sequence Listing.

[0008] The present invention further provides a protein comprising anamino acid sequence having deletion, substitution, addition or insertionof one or more amino acids relative to the amino acid sequence set forthas SEQ ID NO: 2 or NO: 4 in the Sequence Listing and which, whenexpressed in a cell, functions as Na⁺-driven Cl—/HCO₃— exchanger.

[0009] The present invention further provides an above protein whereinthe Na⁺-driven Cl—/HCO₃— exchanger, dependently upon both ofextracellular bicarbonate and intracellular chloride ions, takes upextracellular sodium ion into the cell and transport intracellularsodium ion out of the cell.

[0010] The present invention further provides a cell in which one of theabove proteins is expressed, wherein the cell is of a species differentfrom the species of origin of the one of the proteins. Non-limitingexamples of such cells of different species include Xenopus laevisoocytes and HEK293 cells. Expression of a Na⁺-driven Cl—/HCO₃— exchangerin such cells of different species may be achieved by transfection of aDNA encoding the Na⁺-driven Cl—/HCO₃— exchanger or by introduction of acRNA corresponding to the Na⁺-driven Cl—/HCO₃— exchanger.

[0011] The present invention further provides antibodies to the aboveproteins. The antibodies may be monoclonal or polyclonal.

[0012] The present invention further provides a method for selection ofagonists and antagonists of Na⁺-driven Cl—/HCO₃— exchanger, which methodcomprises bringing a cell of a different species expressing the proteininto contact with a candidate compound, measuring the function of theNa⁺-driven Cl—/HCO₃— exchanger, comparing the result thus obtained witha result obtained by measuring the function of the sodium ion-drivenchloride/bicarbonate exchanger of the cell which has not been broughtinto contact with the candidate compound, and thereby determiningwhether or not the candidate compound enhances or inhibits the function.

[0013] The present invention further provides a DNA comprising thenucleotide sequence set forth as SEQ ID NO: 1 or NO: 3 in the SequenceListing, a DNA >2 comprising a nucleotide sequence consisting ofnucleotides 67 through 3330 in the nucleotide sequence set forth as SEQID NO: 1 in the Sequence Listing, and a DNA comprising a nucleotidesequence consisting of the nucleotides 83 through 3346 in the nucleotidesequence set forth as SEQ ID NO: 3 in the Sequence Listing.

[0014] The present invention further provides a DNA comprising anucleotide sequence having deletion, substitution, addition or insertionof one or more nucleotides relative to a DNA comprising a nucleotidesequence consisting of the nucleotides 67 through 3330 in the nucleotidesequence set forth as SEQ ID NO: 1 in the Sequence Listing, andencoding:

[0015] (1) a protein comprising the amino acid sequence set forth as SEQID NO: 2 in the Sequence Listing, or

[0016] (2) a protein comprising an amino acid sequence having deletion,substitution, addition or insertion of one or more amino acids relativeto the amino acid sequence set forth as SEQ ID NO: 2 in the SequenceListing, which protein, when expressed in a cell, functions asNa⁺-driven Cl—/HCO₃— exchanger.

[0017] The present invention still further provides a DNA comprising anucleotide sequence having deletion, substitution, addition or insertionof one or more nucleotides relative to a DNA comprising a nucleotidesequence consisting of the nucleotides 83 through 3346 in the nucleotidesequence set forth as SEQ ID NO: 3 in the Sequence Listing, andencoding:

[0018] (1) a protein comprising the amino acid sequence set forth as SEQID NO: 4 in the Sequence Listing, or

[0019] (2) a protein comprising an amino acid sequence having deletion,substitution, addition or insertion of one or more amino acids relativeto the amino acid sequence set forth as SEQ ID NO: 4 in the SequenceListing, which protein, when expressed in a cell, functions asNa⁺-driven Cl—/HCO₃— exchanger.

BRIEF DESCRIPTION OF THE FIGURES

[0020]FIG. 1 shows RNA blot analysis of NCBE mRNA in rat tissues andhormone-secreting cell lines (a) and RT-PCR detection of NCBE mRNA frommouse pancreatic islets (b).

[0021]FIG. 2 shows a graph illustrating the effect of extracellularNa⁺concentration on ²²Na⁺uptake.

[0022]FIG. 3 shows a graph illustrating the effect of extracellular HCO₃concentration on ²²Na⁺uptake.

[0023]FIG. 4 shows a graph illustrating the effect of intracellular Cl—on ³⁶Cl— efflux.

[0024]FIG. 5 shows a graph illustrating the effect of DIDS on²²Na⁺uptake.

[0025]FIG. 6 shows a graph illustrating the change in the intracellularpH in the presence and absence of 300 μM DIDS, along with the change inthe intracellular pH in control (non-transfected) cells.

[0026]FIG. 7 shows a graph illustrating the change observed in theintracellular pH when the environment is switched from a Na⁺-freesolution to a Na⁺-containing solution, under a HCO_(3—)-free condition.

[0027]FIG. 8 shows a graph illustrating the change observed in theintracellular pH when the environment is switched from a Na⁺-freesolution to a Na⁺-containing solution, under a Cl—-free condition.

DETAILED DESCRIPTION OF THE INVENTION

[0028] In the present invention, the cells of different species in whichthe protein of the present invention is expressed may be, for example,Xenopus laevis oocytes or HEK293 cells, and selected according to agiven purpose from a variety of cells other than those from mouse orhuman. A conventional method well known in the art may be used forbringing about expression of a protein of the present invention in cellsof species different from the species of origin of the protein.

[0029] In the present specification, the term “one or more” when used inthe context of “an amino acid sequence having deletion, substitution,addition or insertion of one or more amino acids” means a number of oneto ten in general, and preferably a number of one to a few (e.g., threeor four).

[0030] Also in the present specification, the term “one or more” whenused in the context of “a DNA comprising a nucleotide sequence havingdeletion, substitution, addition or insertion of one or morenucleotides” means a number of one to ten in general, and preferably anumber of one to a few (e.g., three or four).

[0031] A variety of such mutant DNAs, as well as mutant proteins encodedby the DNAs, can be produced by means of recombinant DNA technology.First, mutations can be introduced into a cloned DNA fragment throughany of different chemical or enzymatic processes. Mutant DNAs thusobtained are then sequenced for selection of particular mutants withintended merits. This method allows systematic preparation of differentmutants regardless of their phenotypes. General methods for preparingmutant clones are as follows.

[0032] 1. With the help of an oligonucleotide, substitution, deletion,insertion or addition of one or more nucleotides can be directly inducedin a given DNA sequence. This method allows introduction of a number ofmutations into a small region of a given DNA.

[0033] 2. By using a relatively long oligonucleotide, a desired gene canbe synthesized.

[0034] 3. By means of region-specific mutagenesis, a desired mutationcan be introduced into a large (1-3 kb) DNA region.

[0035] 4. Linker-scanning mutagenesis of DNA is a method suitable tointroduce a cluster point mutation into a relatively small (4-10 bp) DNAregion.

[0036] 5. PCR is also utilized as a method for directly introducing amutation. [References: Current protocols in molecular biology. 3 vols.,Edited by Ausubel F. M. et al., John Wiley & Sons, Inc., CurrentProtocols., Vol. 1, Chapter 8: Mutagenesis of cloned DNA, pages8.0.1-8.5.10]

[0037] Also well known to those skilled in the art are methods forpreparing plasmids or other vectors which can express a desired geneincluding different mutations obtained by the above methods. That is, byinserting a DNA comprising a desired gene into an expression vector DNAusing a combination of restriction enzymes and a ligase, a recombinantplasmid is readily constructed which carries the desired gene. Therecombinant plasmid thus obtained is then introduced into differentcells to effect transfection, thereby producing transformed cells. Arange of cells may be utilized, from prokaryotic cells, e.g. E. coli, toyeast, insect, plant and animal cells.

[0038] [Reference: Vectors essential data. Gacesa P. and Ramji D. P. 166pages. BIOS Scientific Publishers Limited 1994., John Wiley & Sons inassociation with BIOS Scientific Publishers Ltd. Expression vectors,pages 9-12.]

[0039] Introduction of a recombinant plasmid into host cells may becarried out by calcium chloride method or by electroporation. Calciumchloride method is an efficient way for achieving transformation and itdoes not requires any apparatus specially designed for it. If stillhigher efficiency is needed, electroporation is recommended.

[0040] [References: Current Protocols in Molecular Biology, 3 Vols.Edited by Ausbel F. M. et al., John Wiley & Sons, Inc., CurrentProtocols, Vol. 1, unit 1.8: Introduction of Plasmid DNA into Cells,pages 1.8.1-1.8.10]

[0041] There are known two types of transfection generally carried outon animal cell lines, i.e., a transient type and a stable and permanenttype. In transient transfection, transformed cells are cultured for 1-4days to allow transcription and replication of the introduced gene, andthen the cells are harvested and their DNA analyzed. In many studies,alternatively, a stable transformant cell line is produced, in which theintroduced gene is incorporated into the chromosomes. Examples of themethod for transfection include calcium phosphate method,electroporation, and liposome fusion method. [Reference: Currentprotocols in molecular biology. 3 vols. Edited by Ausubel F. M. et al.,John Wiley & Son, Inc., Current Protocols. Vol. 1, chapter 9:Introduction of DNA into mammalian cells, pages 9.0.1-9.17.3.]

[0042] Polyclonal and monoclonal antibodies to the Na⁺-driven Cl—/HCO₃—exchanger proteins of the present invention, or to their fragments ortheir analogues, are readily prepared using technologies well known inthe art. Antibodies thus obtained may be used, for example, inimmunohistochemistry of Na⁺-driven Cl—/HCO₃— exchanger protein expressedin cells of different species or for inhibition of its function byblocking the protein. Cells of different species in which the functionof Na⁺-driven Cl—/HCO₃— exchanger is inhibited are used as a control inselection of agonists/antagonists of the protein.

[0043] A general method for preparing a monoclonal antibody in mg-scaledirected to the Na⁺-driven Cl—/HCO₃— exchanger proteins of the presentinvention is as follows: Mice are inoculated with one of the antigenproteins to immunize. The spleen is removed from the mice exhibiting asufficient antibody titer. The spleen cells are dissociated and B cellsare selected and fused with myeloma cells of B cell origin to formhybridoma cells secreting the antibody. The monoclonal antibody secretedby the hybridoma cells is purified from the culture medium by using anaffinity column, or by ion-exchange or gel filtration, etc. Polyclonalantibody of the present invention may also be prepared by a conventionalmethod: using rabbits, horses, mice or guinea pigs as immunized animals,the antigen protein is inoculated along one of the schedules known inthe art to immunize the animals, and then an immunoglobulin such as IgGis isolated from the collected serum.

[0044] [Reference: Current protocols in molecular biology, 3 vols.Edited by Ausubel F. M. et al., John Wiley & Sons, Inc., CurrentProtocols, Vol. 2, chapter 11: Immunology, pages 11.0.1-11.16.13.]

EXAMPLES

[0045] The present invention is described in further details withreference to examples. However, it is not intended that the presentinvention be limited to the examples.

[0046] To determine its structure and functional role, the presentinventors cloned a Na⁺-driven Cl—/HCO₃— exchanger (designated NCBE) fromcDNA library from MIN6, an insulin secreting mouse cell line. Theprimary structure, tissue distribution and functional characterizationof Na⁺-driven chloride (Cl—)/bicarbonate (HCO₃—) exchanger (NCBE) willbe described below.

[0047] It was revealed that the mouse NCBE protein (SEQ ID NO: 2)consists of 1,088 amino acids and has 65, 65 and 41% amino acid identityto the sodium bicarbonate cotransporter from human muscle, retina andkidney, respectively. The mouse NCBE has was found to have ten putativemembrane spanning regions and the conserved4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS)-binding motifcharacteristic of anion exchangers and sodium bicarbonatecotransporters. NCBE mRNA is was shown to be expressed at high levels inthe brain and in a mouse insulinoma cell line MIN6, and, though at lowlevels, also in pituitary, testis, kidney, and ileum. Through functionalanalysis of NCBE protein expressed in Xenopus laevis oocytes and HEK293cells, it was demonstrated that the protein causes a rise inintracellular pH by transporting extracellular Na⁺and HCO₃— into cellsin exchange for intracellular Cl—. Based on the findings, the presentinventors concluded that the cloned NCBE is the Na⁺-driven Cl—/HCO₃—exchanger that regulates intracellular pH in native cells.

[0048] Then, to also identify a human NCBE, a partial sequence (2,746bp) of the mouse Na⁺-driven Cl—/HCO₃— exchanger cDNA obtained above wasfirst amplified by PCR. For this amplification, a DNA fragment havingthe sequence consisting of the nucleotides 250-270 of the sequence setforth as SEQ ID NO: 1 in the Sequence Listing was used as a senseprimer, and, as an antisense primer, a DNA fragment having a sequencecomplementary to the sequence consisting of the nucleotides 2976-2995 ofthe sequence set forth as SEQ ID NO: 1 in the Sequence Listing. PCRconditions were as follows:

[0049] Initial denaturation:94° C., 2 min

[0050] Amplification (20 cycles)

[0051] denaturation:94° C., 15 sec

[0052] annealing:60° C., 30 sec

[0053] extension:72° C., 2 min

[0054] Final extension:72° C., 7 min

[0055] The PCR product thus obtained was labeled with ³²P-dCTP by nicktranslation and used to screen about 1 million phages from a human fetalbrain cDNA library (Clontech). Four positive phage clones were obtainedand their DNAs were digested with EcoRI. After agarose electrophoresis,corresponding bands were excised, and respective DNAs extracted toobtain inserts. Separately, pGEM7Z (Promega) was digested with EcoRI andtreated with alkaline phosphatase. To this, the inserts obtained fromthe positive phages were ligated, respectively, for subcloning. Therespective inserts were then sequenced on an autosequencer (ABI 310),and, based on the sequences thus obtained, the cDNA nucleotide sequencecorresponding to human NCBE protein was determined (set forth as SEQ IDNO: 3). According to the result, the sequence of human NCBE protein thenwas determined (set forth as SEQ ID NO: 4 in the Sequence Listing).

[0056] The methods and results of the above experiments will bedescribed below, focusing on the procedures followed and resultsobtained with mouse NCBE.

[0057] [Materials and Methods]

[0058] cDNA Cloning

[0059] A partial cDNA fragment of human kidney NBC cDNA [Burnham, C. E.,et al., J. Biol. Chem., 272:19111-19114(1997)] amplified by PCR, using ahuman kidney cDNA as a template. The sense and antisense primers used inthis were 5′-TTTGGAGAAAACCCCTGGT-3′ (nt 2232-2250) (SEQ ID NO: 5) and5′-TGACATCATCCAGGAAGCTG-3′ (nt 2912-2931) (SEQ ID NO: 6). PCR wasperformed up to 40 cycles under the following conditions: denaturationat 94° C. for 15 sec, annealing at 60° C. for 30 sec, and extension at72° C. for 45 sec in a thermal cycler GeneAmp PCR system 9600 (PEApplied Biosystems, Foster, Calif.). The 700 bp-PCR product wassubjected to screening of a MIN6 cDNA library [Inagaki, N., et al.,Proc. Natl. Acad. Sci. USA, 91:2679-2683(1994)] as a probe under a lowstringent condition previously described [Fukumoto, H. et al., Proc.Natl. Acad. Sci. USA, 85:5434-5438(1988)]. Positive clones weresubcloned in pGEM-3Z vector (Promega, Madison, WI) and sequenced in bothdirections using ABI PRISM ™ 377 DNA sequencer (PE Applied Biosystems).

[0060] RNA blot analysis

[0061] RNA blot analysis was performed using 10 μg of total RNA fromvarious tissues and cells. The RNAs were denatured with formaldehyde,electrophoresed on 1% agarose gel, and transferred onto a nylonmembrane. The blots were probed with NCBE cDNA under a standardcondition previously described [Wang, C-Z. et al., Biochem. Biophys.Res. Commun., 220: 196-202(1996)]. Before autoradiography, the blotswere washed with 0.1×SSC and 0.1% SDS at room temperature for one hr andthen at 50° C. for another hour.

[0062] Reverse Transcription Polymerase Chain Reaction (RT-PCR)

[0063] Total RNA was prepared from isolated mouse pancreatic islets withTRIZOL Reagent (Life Technologies, Inc., Rockvill, Md.). First-strandcDNA (10 ng) was generated using Superscript™ II reverse transcriptase(Life Technologies) with random primers. PCR was performed with ExpandHigh Fidelity PCR System (Roch Diagnostics, Mannheim, Germany) usingabout 1 ng of template DNA in a 20 μl reaction volume under a standardcondition. The sense and antisense primers used were5′-GTCATGTTAGACCAACAGGT-3′ (nt 4283-4302) (SEQ ID NO: 7) and5′-GTTGTAATAGCGACACTC-3′ (nt 4911-4928) (SEQ ID NO: 8). The PCR productwas resolved on 1% agarose gel and confirmed by DNA sequencing.

[0064] Functional Analysis of NCBE in Xenopus laevis oocytes

[0065] The coding sequence of NCBE in pSD5 was linearized by digestionwith FspI and in vitro transcribed with SP6 RNA polymerase as previouslydescribed (Wang, C-Z. et al., Biochem. Biophys. Res. Commun.,220:196-202(1996)). Defolliculated oocytes were injected with NCBE cRNA(50 nl, 0.5 μg/μl)or water and incubated in 1×MBS medium (88 mM NaCl, 1mM KCl, 0.8 mM MgCl₂, 0.4 mM CaCl₂, 0.3 mM Ca(NO₃)₂, 2.4 mM NaHCO₃ and7.5 mM Tris, pH 7.4) for 3-5 days at 18° C. before the studies. Theoocytes were preincubated for one hr at 18° C. in the standard solution(100 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, and 8 mM NaHCO₃, pH7.4).

[0066] For studies of dependency on extracellular Na⁺concentration, theoocytes were then incubated in 1.4 ml of either 1, 10, 30 or 100 mMNa⁺solution bubbled with 1.5% CO₂, pH 7.4 with 0.074 MBq of ²²Na+ (NEN ™Life Science Products, Boston, Mass.). In each solution, the Na⁺in thestandard solution was substituted with an equal molar amount of choline.A ten μl aliquot was removed from the incubation solution for laterdetermination of ²²Na⁺-specific activity. After 15 min, ²²Na⁺uptake wasterminated by three washes with an ice-cold solution containing 1, 10,30 or 100 mM Na⁺, pH 7.4, respectively, and the oocytes were then lyzedin 0.5 ml of 5% SDS and 4.5 ml of Aqueous Counting Scintillant (AmershamPharmacia Biotech) was added. ²²Na⁺uptake was performed in eitherCl—-free 1, 10, 30 or 100 mM Na⁺solution (pH 7.4). Extracellular Cl— wassubstituted with an equal molar amount of gluconic acid, andextracellular Na⁺was substituted with an equal molar amount ofN-methyl-D-glucamine (NMG). The ²²Na⁺uptake for 15 min was also examinedin the presence or absence of 300 μM4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS, Sigma), aninhibitor of anion-transporters in the standard solution.

[0067] For the study of dependency on extracellular HCO₃— concentration,Na⁺uptake experiments were performed in 1, 3, 10 or 30 mM HCO₃—solutions bubbled with 1.5% CO₂ at 18° C., pH 7.4, including 0.074 MBqof ²²Na⁺. The solutions contained 2 mM KCl, 1 mM MgCl₂ and 1 mM CaCl₂,pH 7.4, and further 107 mM NaCl and 1 mM NaHCO₃ for 1 mM HCO₃— solution,105 mM NaCl and 3 mM NaHCO₃ for 3 mM HCO₃— solution, 98 mM NaCl and 10mM NaHCO₃ for 10 mM HCO₃— solution, and 78 mM NaCl and 30 mM NaHCO₃ for30 mM HCO₃— solution.

[0068] For ³⁶Cl— efflux experiment, the oocytes were preincubated forone hour in the Cl—-free solution for depletion of intracellular Cl—, orCl— containing standard solution. The oocytes were incubated in 0.074MBq of ³⁶Cl—-containing solution (NEN ™ Life Science Products) at 18° C.for one hour bubbling with 1.5% CO₂. The oocytes were rapidly washedthree times with the corresponding, respective solutions and thentransferred into 1.5 ml of each a Cl—-free solution bubbled with 1.5%CO₂, pH 7.4. A 10-μl aliquot was removed from the incubation solutionfor later determination of ³⁶Cl— specific activity. ³⁶Cl— activities inthe solution were measured at 0, 5, 15, 25 and 35 min. The oocytes weretreated as described above for the measurement of the remainingintracellular ³⁶Cl—. Portions of the medium from respective time pointswere counted and the values were summed to determine flux. ³⁶C— effluxwas presented as a percent relative to the total cellular ³⁶Cl—released. ²²Na⁺and ³⁶Cl— activities were measured with betascintillation counter (Aloka, Japan).

[0069] Functional Analysis of NCBE in HEK293 Cells

[0070] HEK293 cells were plated at a density of 3×10⁵ cells per 3.5cm-diameter dish containing a coverslip, and cultured in Dulbecco'smodified Eagle's medium (DMEM, high glucose) supplemented with 10% fetalbovine serum, streptomycin (60.5 μg/ml), and penicillin (100 μg/ml) at37° C. under a humidified condition of 95% air and 5% CO₂. Cells weretransfected with 1 μg of the full-length NCBE cDNA in the pcDNA3.1vector (Invitrogen, Groningen, The Netherlands) using Lipofectamine,Lipofectamine Plus, and Opti-MEM I reagents (Life Technologies,Gaithersburg, Md.) according to the manufacturer's instructions. Thecells were studied 48-72 hours after transfection. Changes inintracellular pH were monitored using2′,7′-bis-(2-carboxyethyl)-5-(6)-carboxyfluorescein, acetoxymethyl ester(BCECF-AM, Molecular Probe, Eugene, OR) (Burnham, C. E., et al., J.Biol. Chem., 272:19111-19114(1997)). HEK293 cells were loaded with 1 μMBCECF-AM for one hour and monitored for changes in intracellular pH bydual-excitation wavelength method with a computerized image processor(490 nm/450 nm; 520-560 nm emission) (Argus-50; Hamamatsu Photonics,Hamamatsu, Japan). ΔpH_(i) was determined as the difference between theintracellular pH before and 10 min after switching to the test solution.The pH_(i) calibration curve was generated using KCl/nigericin technique(Thomas, J. A. et al., Biochemistry 18:2210-2218(1979)). In all theexperiments, the cells were first acidified by NH₄ ⁺-prepulse with 40 mMNH₄Cl-containing solution for 5 min before switching to theNa⁺-containing respective test solutions (Burnham, C. E., et al., J.Biol. Chem., 272:19111-19114(1997)).

[0071] To estimate Na⁺-dependency of the intracellular pH (ΔpH^(i))recovery from intracellular acidification, a Na⁺-free solution (115 mMtetramethylammonium chloride (TMA-Cl), 25 mM KHCO₃, 0.8 mM K₂HPO₄, 0.2mM KH₂PO₄, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES) and a Na⁺-containingsolution (TMA-Cl and KHCO₃ in the Na⁺-free solution were replaced with90 mM NaCl, 25 mM KCl, and 25 mM NaHCO₃) were used.

[0072] To test for HCO—-dependency, a HCO₃—-free, Na⁺-free solution (115mM TMA-Cl, 0.8 mM K₂HPO₄, 0.2 mM KH₂PO₄, 1 mM CaCl₂, 1 mM MgCl₂, 10 mMHEPES) and a HCO₃—-free, Na⁺-containing solution (in which TMA-Cl in theHCO₃—-free, Na⁺-free solution was replaced with 90 mM NaCl and 25 mMKCl) were used.

[0073] To determine Cl—-dependency, a Cl—-free, Na⁺-free solution (25 mMKHCO₃, 0.8 mM K₂HPO₄, 0.2 mM KH₂PO₄, 10 mM HEPES, 115 mM NMG-gluconate)and a Cl—-free, Na⁺-containing solution (in which NMG-gluconate wasreplaced with 115 mM sodium gluconate) were used and the results werecompared with each other.

[0074] All the solutions were bubbled with 95% O₂ and 5% CO₂, and theirpH adjusted to 7.4. The osmolarity of each solution was adjusted withsucrose. The assays were carried out at 37° C.

[0075] Statistical Analysis

[0076] The results were expressed as means ± SE. Statisticalsignificance between experiments was determined by Student's t test.

[0077] [Results and Discussion]

[0078] NCBE is structurally related to Na⁺—HCO₃— transporters.

[0079] As described above, the cDNA encoding Na⁺-driven Cl—/HCO₃—exchanger (NCBE) was cloned from a MIN6 cDNA by screening it using apartial human kidney Na⁺—HCO₃— cotransporter (NBC) cDNA as a probe. Thethus determined nucleotide sequence (NCBE) is set forth as SEQ ID NO: 1in the Sequence Listing. The composite 5,385-bp nucleotide sequencecontains an open reading frame, which follows an in-frame terminationsignal upstream of the “ATG” and encodes a protein of 1,088 amino acidsset forth as SEQ ID NO: 1 having a predicted molecular weight of 122kDa. A hydrophobicity analysis indicates that the amino acid sequencehas putative membrane spanning segments (TM1 to TM10) at the followingpositions, respectively.

[0080] TM 1: amino acids 479˜499

[0081] TM2: amino acids 514˜534

[0082] TM3: amino acids 564˜584

[0083] TM4: amino acids 693˜713

[0084] TM5: amino acids 733˜753

[0085] TM6: amino acids 780˜800

[0086] TM7: amino acids 826˜846

[0087] TM8: amino acids 882˜901

[0088] TM9: amino acids 905˜924

[0089] TM10: amino acids 972˜992

[0090] In the amino acid sequence, there are three potential N-linkedglycosylation sites in the extracellular loops between the third (TM3)and fourth (TM4) spanning region (Asn-647, Asn-657 and Asn-667).Putative DIDS-binding motif is at amino acids 815-818.

[0091] Comparison of amino acid sequence between NCBE and other NBCsshowed that NCBE has 65%, 65% and 41% amino acid identity to humanmuscle NBC [Pushkin, A. et al., J. Biol. Chem., 274:16569-16575(1999)],human retina NBC [Ishibashi, K. et al., Biochem. Biophys. Res. Commun.,24:535-538(1998)], and human kidney NBC [Burnham, C. E., et al., J.Biol. Chem., 272:19111-19114(1997)], respectively. This indicates thatNCBE represents a novel bicarbonate transporter. The amino acidsequences in the putative transmembrane regions and DIDS-binding motifLys Leu Lys Lys (residue 815-818) are well conserved in NCBE, whilethose in the intracellular amino- and carboxyl-terminal regions and inthe large extracellular loop between the third and the fourth membranespanning regions are rather divergent.

[0092] NCBE is expressed at high levels in the brain andinsulin-secreting clonal pancreatic β-cells.

[0093] RNA blot analysis revealed a 5.5 kb NCBE mRNA is expressed athigh levels in brain and the insulin secreting cell line MIN6 cells andexpressed at low levels in pituitary, testis, kidney, and ileum (FIG. 1,a). RT-PCR analysis shows that NCBE is also expressed in pancreaticislets (FIG. 1, b).

[0094] In the figure, “a” represents the result of the RNA blot analysisof NCBE mRNA in rat tissues and hormone-secreting cell lines. The sizeof hybridized transcripts is indicated. “b” represents the results ofRT-PCR detection of NCBE mRNA in mouse pancreatic islets. DNA lengthmarkers and RT-PCR products are shown in lanes 1 and 2, respectively.

[0095] NCBE is a Na⁺-driven Cl—/HCO₃— exchanger that regulatesintracellular pH (pH_(i)).

[0096] The present inventors examined the functional properties of NCBEusing Xenopus laevis oocyte system. ²²Na⁺uptake and ³⁶Cl— efflux weremeasured 3-5 days after injection of the cRNAs or water (control).Bubbling with 1.5% CO₂ to acidify the oocytes, the present inventorsfirst examined the effect of extracellular Na⁺concentration on²²Na⁺uptake. The results are shown in FIG. 3.

[0097]FIG. 3 illustrates the relation between ²²Na⁺uptake(nmol/oocyte/hour) and extracellular Na⁺concentration. In the figure, ▪and  indicate the results obtained with the cells injected with NCBEcRNA, and □ and ◯ the results obtained with the cells injected withwater. ▪ and □ indicate the results obtained using Cl—-containingextracellular solutions, and  and ◯ indicate the results obtained usingCl—-free extracellular solutions. The respective data represent the mean±SE (standard error) for 7 to 16 oocytes from two independentexperiments. * and † (p<0.05) indicate the presence/absence ofstatistical significance in the difference from water-injected cells andfrom incubation in Cl—-free extracellular solutions, respectively, with10, 30 or 100 mM Na⁺.

[0098] As shown in FIG. 2, the increase in ²²Na⁺uptake was dependent onextracellular Na⁺concentrations, with a linear pattern observed in NCBEcRNA-injected oocytes over the physiological range of Na⁺concentrations.The water-injected oocytes showed no increase in ²²Na⁺uptake. Comparisonof Na⁺uptake between the results obtained with Cl—-containing andCl—-free solutions showed significantly higher Na⁺uptake in the presenceof extracellular Cl— than the in the absence of extracellular Cl— (FIG.2). These results indicate that NCBE transports extracellular Na⁺intothe cells and that extracellular Cl— participates in acceleration of theNCBE's activity.

[0099] The present inventors, then, examined the effect of extracellularbicarbonate ion on ²²Na⁺uptake. The results are shown in FIG. 3. Therespective data represent the mean±SE (standard error) for 11 to 16oocytes from two independent experiments. * (p<0.05) indicatescomparison with water-injected cells. As evident from the figure,increased extracellular bicarbonate ion significantly boosted Na⁺uptakein a concentration-dependent manner in the NCBE cRNA-injected oocytes,while the water-injected oocytes did not show any such change inNa⁺uptake. These results indicate that extracellular bicarbonate ion isnecessary in transporting Na⁺into the cells.

[0100] To determine whether Cl— is transported into or out of the cellsby NCBE, the present inventors examined ³⁶Cl— efflux from Xenopus laevisoocytes. As ³⁶Cl— influx was not detected in water-injected oocytes,analysis was made only for ³⁶Cl— efflux from NCBE cRNA-injected oocytes.The rate (%) of ³⁶Cl— efflux from NCBE cRNA-injected oocytes wasmeasured from 0 to 35 min under the intracellular Cl—-depleted conditionby preincubation with a Cl—-free solution and under the intracellularCl— non-depleted condition by preincubation with Cl—-containingsolution. The results are shown in FIG. 4. In the figure,  indicatesthe results obtained with cells under the intracellular Cl— non-depletedcondition (preincubation in the Cl—-containing solution), and ▴indicates the results obtained with cells under the intracellularCl—-depleted condition (preincubation in the Cl—-free solution). Thedata represent the mean ±SE (standard error) for 16 to 17 oocytes fromthree independent experiments. * (p<0.05) indicates comparison withintracellular Cl—-depleted cells, at 5, 15, 25, and 35 min.

[0101] Comparison made among results of ³⁶Cl— efflux under the differentconditions indicates that NCBE transports intracellular Cl— out of thecells. Taken together, these results demonstrate that NCBE exchangesextracellular Na⁺and bicarbonate ion with intracellular Cl—.

[0102] The present inventors also examined the effect of DIDS, aninhibitor of anion-transporter, on ²²Na⁺uptake. Expression was assessedin the absence or presence of 0.3 mM DIDS. The results are shown in FIG.5. The data represent the mean ±SE (standard error) for 21 to 22 oocytesfrom three independent experiments. * (p<0.05) indicates comparison withcRNA+DIDS.

[0103] While the ²²Na⁺uptake in NCBE cRNA-injected oocytes was 31.4±2.1nmol/oocyte/hour (n=21) in the absence of DIDS, it was 6.0±0.7nmol/oocyte/hour (n=14) in the presence of 300 μM DIDS. Inwater-injected oocytes, the uptake was 1.6±0.3 (n =22) and 2.1±0.4(n=19) nmol/oocytes/hour in the absence and presence of DIDS,respectively. Thus, DIDS was shown to partially inhibit ²²Na⁺uptake byNCBE (FIG. 5).

[0104] To clarify the role of NCBE in the regulation of intracellularpH, changes in intracellular pH were measured under various conditionsusing HEK293 cells transiently transfected with NCBE. All theexperiments were performed under conditions where the intracellular pHwas acidified with NH₄+prepulse. To determine whether the change in theintracellular pH is dependent on extracellular Na⁺, the environment ofthe cells was switched from a Na⁺-free solution to a Na⁺-containingsolution. The results are shown in FIG. 6. FIG. 6 is a graph illustratesa trace of control (non-transfected) cells and NCBE-transfected cellswith or without 300 μM DIDS. The environment of the cells was switchedfrom a Na⁺-free solution to a Na⁺-containing solution.

[0105] As shown in the figure, a rapid recovery of intracellular pH(ΔpH_(i)) was observed only in the NCBE-transfected cells in thepresence of 1 mM 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), a specificinhibitor of Na⁺/H+exchanger (ΔpH_(i) was 0.239±0.028 (n=97) in theNCBE-transfected cells and 0.003±0.015 (n=70) in the control. p<0.05)(FIG. 6). This recovery in intracellular pH was partially inhibited by300 μM DIDS (ΔpH_(i) was 0.023±0.042 (n=89). p<0.05).

[0106] To determine whether this change in intracellular pH isbicarbonate ion-dependent, the environment of the NCBE-transfected cellswas switched from a HCO₃—-free, Na⁺-free solution to a HCO₃—-free butNa⁺-containing solution, in the presence of 1 mM EIPA. However, as shownin FIG. 7, no recovery of intracellular pH was detected (ΔpH_(i) was0.002±0.014 (n=71)).

[0107] Finally, an examination for Cl— dependency was also made by thepresent inventors. NCBE-transfected cells were kept in a Cl—-freesolution (under an intracellular Cl—-depletion condition) throughout theexperiments. Under this condition, the environment of the cells wasswitched from a Na⁺-free solution to a Na⁺-containing solution. In thepresence of 1 mM EIPA, as shown in FIG. 8, no recovery of intracellularpH was detected [ΔpH, was 0.067±0.012 (n=95)].

[0108] These results indicate that recovery of intracellular pH fromintracellular acidification is detected only where extracellular Na⁺andHCO₃— and intracellular Cl— are present.

[0109] The studies of the function of NCBE heterologously expressed inXenopus laevis oocytes and HEK293 cells show that NCBE allowsintracellular pH to recover from acute intracellular acidification, bytransporting extracellular Na⁺and HCO₃— in exchange for intracellularCl— (FIGS. 3 and 4). NCBE is functionally distinct from so far reportedanion exchangers and Na⁺—HCO₃— cotransporters. This is because:1) NCBE,expressed in Xenopus laevis, exhibited a Na⁺uptake increase dependent onintracellular Cl—, 2) it shows the ability of exporting Cl— out of thecells, and, furthermore, 3) the NCBE, expressed in HEK239 cells,elevates intracellular pH in a manner dependent upon extracellularNa⁺and HCO₃—, and intracellular Cl—. These properties are similar tothose of Na⁺-driven Cl—/HCO₃— exchanger described in native cells. Thecloned NCBE, therefore, is concluded to be a Na⁺-driven Cl—/HCO₃—exchanger.

[0110] Possible physiological relevance of NCBE. That NCBE mRNA isexpressed in insulin secreting cell line MIN6 and pancreatic isletsimplies its physiological relevance. It has been shown thatglucose-induced insulin secretion is accompanied by a rise inintracellular pH in pancreatic β-cells. While several intracellular pHregulators have been suggested to be present in pancreatic β-cells,their molecular basis has not been known so far. NCBE is the firstintracellular pH-regulating exchanger whose primary structure andfunctional properties have been determined. NCBE most likely contributesto the process for recovery of intracellular pH in pancreatic β-cellsthat have been acidified by glucose metabolism. NCBE mRNA occurs also inthe testis, although its expression level is low. It has been shown thatintracellular pH regulates many functions in sperm including spermcapacitation. As sperm capacitation results in the increase inintracellular pH, which requires functional Na⁺, Cl— and HCO₃—-dependentacid-efflux pathway, NCBE could participate in the process of spermcapacitation. NCBE mRNA is also expressed at high levels in the brain.Though physiological studies suggests that NCBE is present inhippocampal neurons and astrocytes, its physiological significance ofsuch cells remains unknown at present.

1 8 1 5385 DNA Mus musculus 1 ggctgagtgg aagacactga agacactgcagagcaaggtg ctttttttcc agaggtgtta 60 cagaac atg gag att aaa gac cag ggagcc caa atg gag ccg ctg ctg 108 Met Glu Ile Lys Asp Gln Gly Ala Gln MetGlu Pro Leu Leu 1 5 10 cct acg aga aat gat gaa gaa gcc gtt gtg gat agaggt gga aca cgc 156 Pro Thr Arg Asn Asp Glu Glu Ala Val Val Asp Arg GlyGly Thr Arg 15 20 25 30 tct att ctc aaa aca cat ttt gag aaa gaa gat ttagaa ggt cat cgg 204 Ser Ile Leu Lys Thr His Phe Glu Lys Glu Asp Leu GluGly His Arg 35 40 45 aca tta ttt att gga gtt cat gtg ccc ctg ggt gga agaaaa agc cat 252 Thr Leu Phe Ile Gly Val His Val Pro Leu Gly Gly Arg LysSer His 50 55 60 cgt cgt cac agg cat cgt ggt cat aag cac aga aag agg gacaga gag 300 Arg Arg His Arg His Arg Gly His Lys His Arg Lys Arg Asp ArgGlu 65 70 75 aga gat tcg gga ctg gag gat gga aga gag tcc cct tct ttt gacacc 348 Arg Asp Ser Gly Leu Glu Asp Gly Arg Glu Ser Pro Ser Phe Asp Thr80 85 90 cca tcg cag agg gtg cag ttt att ctt gga act gag gac gat gat gag396 Pro Ser Gln Arg Val Gln Phe Ile Leu Gly Thr Glu Asp Asp Asp Glu 95100 105 110 gag cac ctc cct cat gac ctt ttc aca gag ctg gat gag att tgctgg 444 Glu His Leu Pro His Asp Leu Phe Thr Glu Leu Asp Glu Ile Cys Trp115 120 125 cgt gaa ggg gaa gat gct gag tgg cga gag aca gcc agg tgg ttgaaa 492 Arg Glu Gly Glu Asp Ala Glu Trp Arg Glu Thr Ala Arg Trp Leu Lys130 135 140 ttt gaa gag gat gtg gaa gat gga gga gaa aga tgg agt aag ccctat 540 Phe Glu Glu Asp Val Glu Asp Gly Gly Glu Arg Trp Ser Lys Pro Tyr145 150 155 gtg gcc acg ctt tca tta cac agc ttg ttt gag ttg aga agc tgcatc 588 Val Ala Thr Leu Ser Leu His Ser Leu Phe Glu Leu Arg Ser Cys Ile160 165 170 ctg aat gga act gtg cta ctg gac atg cat gcc aac acg ata gaagaa 636 Leu Asn Gly Thr Val Leu Leu Asp Met His Ala Asn Thr Ile Glu Glu175 180 185 190 att gca gat atg gtc ctt gac cag cag gtc agc tca ggc cagctg aat 684 Ile Ala Asp Met Val Leu Asp Gln Gln Val Ser Ser Gly Gln LeuAsn 195 200 205 gaa gat gtt cgc cac agg gtc cac gaa gca ttg atg aag cagcat cat 732 Glu Asp Val Arg His Arg Val His Glu Ala Leu Met Lys Gln HisHis 210 215 220 cac cag aat cag aaa aaa ctg gct aac agg att cct att gtccga tct 780 His Gln Asn Gln Lys Lys Leu Ala Asn Arg Ile Pro Ile Val ArgSer 225 230 235 ttg gct gat att ggc aag aaa caa tca gaa cca aat tcc atggat aaa 828 Leu Ala Asp Ile Gly Lys Lys Gln Ser Glu Pro Asn Ser Met AspLys 240 245 250 aat gca ggt cag gtt gtt tct cct cag tct gct cca gcc tgtgct gag 876 Asn Ala Gly Gln Val Val Ser Pro Gln Ser Ala Pro Ala Cys AlaGlu 255 260 265 270 aat aaa aat gat gtc agc agg gaa aac agc act gta gacttc agc aag 924 Asn Lys Asn Asp Val Ser Arg Glu Asn Ser Thr Val Asp PheSer Lys 275 280 285 gtt gat ctg cat ttt atg aaa aag att cct ccg ggt gctgaa gct tca 972 Val Asp Leu His Phe Met Lys Lys Ile Pro Pro Gly Ala GluAla Ser 290 295 300 aac atc ttg gta gga gaa ctg gag ttc cta gac aga gctgtg gtt gcc 1020 Asn Ile Leu Val Gly Glu Leu Glu Phe Leu Asp Arg Ala ValVal Ala 305 310 315 ttt gtc agg ttg tct cca gct gtc ttg ctc caa gga cttgct gaa gtt 1068 Phe Val Arg Leu Ser Pro Ala Val Leu Leu Gln Gly Leu AlaGlu Val 320 325 330 cca atc cca agc aga ttt ctg ttc atc ctt ctg gga cccctg gga aag 1116 Pro Ile Pro Ser Arg Phe Leu Phe Ile Leu Leu Gly Pro LeuGly Lys 335 340 345 350 ggt caa cag tac cac gag att ggc aga tcg att gcgacc tta atg act 1164 Gly Gln Gln Tyr His Glu Ile Gly Arg Ser Ile Ala ThrLeu Met Thr 355 360 365 gat gag gtg ttt cat gat gtt gct tac aaa gct aaagac cgc aat gac 1212 Asp Glu Val Phe His Asp Val Ala Tyr Lys Ala Lys AspArg Asn Asp 370 375 380 ttg gta tca gga att gat gag ttt ctg gat cag gttacc gtt ctt cct 1260 Leu Val Ser Gly Ile Asp Glu Phe Leu Asp Gln Val ThrVal Leu Pro 385 390 395 cct gga gaa tgg gat cca agc ata cga ata gaa cctccc aaa aat gtc 1308 Pro Gly Glu Trp Asp Pro Ser Ile Arg Ile Glu Pro ProLys Asn Val 400 405 410 cct tcc cag gag aag agg aag att cct gct gta ccaaat gga aca gca 1356 Pro Ser Gln Glu Lys Arg Lys Ile Pro Ala Val Pro AsnGly Thr Ala 415 420 425 430 gct cat ggc gaa gct gag cca cat gga gga cacagc gga cct gaa ctc 1404 Ala His Gly Glu Ala Glu Pro His Gly Gly His SerGly Pro Glu Leu 435 440 445 cag cga act ggg agg att ttt ggg gga ctt atgtta gat atc aaa aga 1452 Gln Arg Thr Gly Arg Ile Phe Gly Gly Leu Met LeuAsp Ile Lys Arg 450 455 460 aag gct cca ttc ttc tgg agc gac ttc agg gatgct ttc agc ctg cag 1500 Lys Ala Pro Phe Phe Trp Ser Asp Phe Arg Asp AlaPhe Ser Leu Gln 465 470 475 tgc tta gca tcg ttc ctg ttt ctc tac tgt gcatgc atg tct cct gtc 1548 Cys Leu Ala Ser Phe Leu Phe Leu Tyr Cys Ala CysMet Ser Pro Val 480 485 490 atc aca ttt gga gga ctg ttg gga gaa gca actgaa ggt cgt ata agt 1596 Ile Thr Phe Gly Gly Leu Leu Gly Glu Ala Thr GluGly Arg Ile Ser 495 500 505 510 gca atc gaa tca ctc ttt gga gca tct atgacc ggg ata gcc tat tct 1644 Ala Ile Glu Ser Leu Phe Gly Ala Ser Met ThrGly Ile Ala Tyr Ser 515 520 525 ctt ttt ggt gga cag ccc ctg acc ata ttaggc agc aca gga cct gtt 1692 Leu Phe Gly Gly Gln Pro Leu Thr Ile Leu GlySer Thr Gly Pro Val 530 535 540 ttg gtg ttt gaa aag atc ttg ttt aag ttttgc aag gaa tac ggc ctg 1740 Leu Val Phe Glu Lys Ile Leu Phe Lys Phe CysLys Glu Tyr Gly Leu 545 550 555 tcg tac ttg tcc tta cgg gcc agc att gggctc tgg act gca aca ctg 1788 Ser Tyr Leu Ser Leu Arg Ala Ser Ile Gly LeuTrp Thr Ala Thr Leu 560 565 570 tgc atc atc ctt gtg gcc acg gac gcg agctca ctc gtc tgc tac atc 1836 Cys Ile Ile Leu Val Ala Thr Asp Ala Ser SerLeu Val Cys Tyr Ile 575 580 585 590 acc cgg ttt acc gaa gag gct ttt gcttct ctc att tgc atc att ttt 1884 Thr Arg Phe Thr Glu Glu Ala Phe Ala SerLeu Ile Cys Ile Ile Phe 595 600 605 atc tat gaa gcc ctg gag aag ttg tttgag ctc agt gaa acc tat cca 1932 Ile Tyr Glu Ala Leu Glu Lys Leu Phe GluLeu Ser Glu Thr Tyr Pro 610 615 620 atc aat atg cac aat gat ttg gaa ctgctg aca caa tac tca tgt aac 1980 Ile Asn Met His Asn Asp Leu Glu Leu LeuThr Gln Tyr Ser Cys Asn 625 630 635 tgt atg gag cca cat agt ccc agc aatgac aca ctg aag gaa tgg cgg 2028 Cys Met Glu Pro His Ser Pro Ser Asn AspThr Leu Lys Glu Trp Arg 640 645 650 gag tcc aac ctt tct gcc tct gac ataatc tgg ggg aac cta act gtg 2076 Glu Ser Asn Leu Ser Ala Ser Asp Ile IleTrp Gly Asn Leu Thr Val 655 660 665 670 tca gag tgc aga tca ctg cac ggggag tat gtc ggg cga gcc tgt ggc 2124 Ser Glu Cys Arg Ser Leu His Gly GluTyr Val Gly Arg Ala Cys Gly 675 680 685 cat ggc cac ccc tac gtg cca gatgtt ctc ttc tgg tcg gtg atc ctg 2172 His Gly His Pro Tyr Val Pro Asp ValLeu Phe Trp Ser Val Ile Leu 690 695 700 ttc ttc tcc aca gtt acc atg tcagcc acc ctg aag cag ttc aag acc 2220 Phe Phe Ser Thr Val Thr Met Ser AlaThr Leu Lys Gln Phe Lys Thr 705 710 715 agc cgc tat ttc cca acc aag gttcga tcc ata gtg agt gat ttt gcg 2268 Ser Arg Tyr Phe Pro Thr Lys Val ArgSer Ile Val Ser Asp Phe Ala 720 725 730 gtt ttt ctt aca att ctg tgt atggtt tta att gac tat gcc att ggg 2316 Val Phe Leu Thr Ile Leu Cys Met ValLeu Ile Asp Tyr Ala Ile Gly 735 740 745 750 atc cca tca cca aaa cta caagta cca agc gtt ttc aag ccg acc ata 2364 Ile Pro Ser Pro Lys Leu Gln ValPro Ser Val Phe Lys Pro Thr Ile 755 760 765 tac gac cgt ggc tgg ttt gttaca cct ttg ggt cca aac cca tgg tgg 2412 Tyr Asp Arg Gly Trp Phe Val ThrPro Leu Gly Pro Asn Pro Trp Trp 770 775 780 aca atc ata gct gcc atc atccca gct tta ctc tgt act att ctg att 2460 Thr Ile Ile Ala Ala Ile Ile ProAla Leu Leu Cys Thr Ile Leu Ile 785 790 795 ttc atg gac cag cag att acagct gtc atc atc aac aga aaa gag cac 2508 Phe Met Asp Gln Gln Ile Thr AlaVal Ile Ile Asn Arg Lys Glu His 800 805 810 aag cta aag aaa ggt tgt ggctat cac ctg gat ctg tta atg gtg gca 2556 Lys Leu Lys Lys Gly Cys Gly TyrHis Leu Asp Leu Leu Met Val Ala 815 820 825 830 gtc atg ctc ggg gtc tgctcc att atg ggc ctg cca tgg ttt gtg gct 2604 Val Met Leu Gly Val Cys SerIle Met Gly Leu Pro Trp Phe Val Ala 835 840 845 gcc aca gtt ctc tcc atcact cat gtc aac agc ctc aag ctc gaa tca 2652 Ala Thr Val Leu Ser Ile ThrHis Val Asn Ser Leu Lys Leu Glu Ser 850 855 860 gag tgc tct gct cca ggagaa caa ccc aag ttt ctc ggc att cgg gag 2700 Glu Cys Ser Ala Pro Gly GluGln Pro Lys Phe Leu Gly Ile Arg Glu 865 870 875 cag agg gtg acc ggg ctcatg att ttt att ctt atg ggt tca tcc gtt 2748 Gln Arg Val Thr Gly Leu MetIle Phe Ile Leu Met Gly Ser Ser Val 880 885 890 ttc atg acc agc att ctgaag ttt atc ccc atg cca gtg tta tac gga 2796 Phe Met Thr Ser Ile Leu LysPhe Ile Pro Met Pro Val Leu Tyr Gly 895 900 905 910 gtg ttt ctt tat atgggt gct tcg tct ctc aaa gga att cag tta ttt 2844 Val Phe Leu Tyr Met GlyAla Ser Ser Leu Lys Gly Ile Gln Leu Phe 915 920 925 gat aga ata aag ctcttc tgg atg cca gcc aaa cat caa cca gat ttc 2892 Asp Arg Ile Lys Leu PheTrp Met Pro Ala Lys His Gln Pro Asp Phe 930 935 940 atc tat cta agg cacgtg ccc ctc cgg aaa gtc cat ctc ttc aca gtc 2940 Ile Tyr Leu Arg His ValPro Leu Arg Lys Val His Leu Phe Thr Val 945 950 955 att cag atg agt tgtctc ggc ctt ctg tgg ata atc aaa gtt tcg aga 2988 Ile Gln Met Ser Cys LeuGly Leu Leu Trp Ile Ile Lys Val Ser Arg 960 965 970 gct gct att gtc tttcct atg atg gtg ttg gca cta gtg ttt gtg aga 3036 Ala Ala Ile Val Phe ProMet Met Val Leu Ala Leu Val Phe Val Arg 975 980 985 990 aag ttg atg gacttc ttg ttt acc aaa cgg gaa ctc agc tgg ctt gat 3084 Lys Leu Met Asp PheLeu Phe Thr Lys Arg Glu Leu Ser Trp Leu Asp 995 1000 1005 gat tta atgcct gag agt aaa aag aag aaa ctt gaa gat gct gag aaa 3132 Asp Leu Met ProGlu Ser Lys Lys Lys Lys Leu Glu Asp Ala Glu Lys 1010 1015 1020 gaa gaagaa caa agt atg cta gcc atg gag gac gag ggc aca gta caa 3180 Glu Glu GluGln Ser Met Leu Ala Met Glu Asp Glu Gly Thr Val Gln 1025 1030 1035 ctccca ctg gag gga cac tac aga gac gac ccg tct gtg atc aat att 3228 Leu ProLeu Glu Gly His Tyr Arg Asp Asp Pro Ser Val Ile Asn Ile 1040 1045 1050tct gat gaa atg tca aag act gcc atg tgg ggg aac ctt cta gtt act 3276 SerAsp Glu Met Ser Lys Thr Ala Met Trp Gly Asn Leu Leu Val Thr 1055 10601065 1070 gct gac aac tca aaa gaa aag gag tca cgc ttt cct tct aaa agctcc 3324 Ala Asp Asn Ser Lys Glu Lys Glu Ser Arg Phe Pro Ser Lys Ser Ser1075 1080 1085 cct tcc taa tcactctaga agctgattcc ccaaagcaat gaaagccgaaaggagaa 3380 Pro Ser gaaagctgac tcagggaaag gcgttgacag ggagacttgtctatgacttg atcttcaatt 3440 tattttttac atatatatat atgagaagag tgtcacaattattaacaaaa ctgctttgat 3500 catgtaattg taaaccctct ctcccatccc accttcatactgtaagtagt gcaagccttc 3560 attctatttc tgtgttcagc ctctgagcag gtcgacacccttgtaagcag atccaatagc 3620 taatgcaaga gtctccagtg ttactgccgt aagacattcgccaacacagg attctcattg 3680 ttgacattaa gagaacaaag ctttctttaa aagataagttatatttgcct agtttgtatt 3740 ttcctacctt agtaacctga agatgcctga taattttattcagaagaatt ttgaaaggta 3800 gtcgtacttt ttatttttta tggcttagca ttcgttactggttttgaaag acccaaatca 3860 aaaagttact ctgaaagcat ttttaataat tgtatttatgtatttccttg acttaatatg 3920 aaacatttaa tacttaataa ctgttacttc aagtcatttgagaaagagac ctgttcatat 3980 cttcttaaaa gacatactgc aaagagtcaa gtagtgttcacttagaattc aagttgtaac 4040 catgcagtca aaaactaggc ttgtattaaa tgctttagagatatttgaag agttttgtgg 4100 ggcttttcat tttaaatctt taccagaaat atgctactgagtttctctcc cattgacaag 4160 ggttgcttcc cgaataagcc tatgacatac atacttacggaatgccacat ggtgcaacat 4220 tgtacatttg atgccagccc tggcagctgt tctgctgaccatggtcatgt gctgctaagt 4280 ttggttccta tcatgttgtc atgttagacc aacaggtctccaactgtatt ttgttttttt 4340 tgcaaagctc ttttccacat tttaactaaa tgcatgttgtggaaaaatag tctttgaaat 4400 aaaatttcag attttgttag aaaaggttat gtaaatacttcagtccatat gaaacagttc 4460 aactttattg aaacaggaag gagattatgg atttttgagtattactaaat ataaatttca 4520 tttaattttc aataaatgtg ctttaataca aaacaaaatatcataggggt cttagttcct 4580 aaaaaagtat caatgattaa caaccttata atctttcaatgtccaggttt agaaaaattc 4640 agagccttct gggttttata aattacatgt actctgtgtaaatacacata attagaaaaa 4700 tcctctttgc ttttaagcta atgaagacga gagacaacagagcctacata accttaatat 4760 tctgatatct tgaacaaaaa atttcctcag aatcctttcaggagccattt ttttaatgag 4820 atatgagcca aaattgtgag aagaattttc agttcgtaaagtctgtattt ataaatggta 4880 aagaaaaatg caaaattctt ttccaaatgt gctacctttgtgatagttgt aatagcgaca 4940 ctctctctaa acattctcgc tgtctatgac ttagcaggccaatccccaaa gcactctcct 5000 ggtgtctcta gagtgtcatg tctgttctgt tgaaatgaccagtgagtgac acttcacatg 5060 atcactggtt taaacaggca atcagcctat gaaattctgtatttctgaat atttttatag 5120 taattttgtt cttgtgtgaa ttttaatgct atctctatcttaatcttaat attttgaaat 5180 cacataaaat ataagaaaat gtagtattct atatttactctaatttcaga ttcctggtca 5240 aaattactga atatcttgaa tgtaatttat tgcaatgtttaagtactgtg taaatgtgac 5300 aggatattgt gtttttcaaa actaagaaat gttatgtggaaataaatatt tatcctaaaa 5360 aaaaaaaaaa aaaaaaaaaa aaaaa 5385 2 1088 PRTMus musculus 2 Met Glu Ile Lys Asp Gln Gly Ala Gln Met Glu Pro Leu LeuPro Thr 1 5 10 15 Arg Asn Asp Glu Glu Ala Val Val Asp Arg Gly Gly ThrArg Ser Ile 20 25 30 Leu Lys Thr His Phe Glu Lys Glu Asp Leu Glu Gly HisArg Thr Leu 35 40 45 Phe Ile Gly Val His Val Pro Leu Gly Gly Arg Lys SerHis Arg Arg 50 55 60 His Arg His Arg Gly His Lys His Arg Lys Arg Asp ArgGlu Arg Asp 65 70 75 80 Ser Gly Leu Glu Asp Gly Arg Glu Ser Pro Ser PheAsp Thr Pro Ser 85 90 95 Gln Arg Val Gln Phe Ile Leu Gly Thr Glu Asp AspAsp Glu Glu His 100 105 110 Leu Pro His Asp Leu Phe Thr Glu Leu Asp GluIle Cys Trp Arg Glu 115 120 125 Gly Glu Asp Ala Glu Trp Arg Glu Thr AlaArg Trp Leu Lys Phe Glu 130 135 140 Glu Asp Val Glu Asp Gly Gly Glu ArgTrp Ser Lys Pro Tyr Val Ala 145 150 155 160 Thr Leu Ser Leu His Ser LeuPhe Glu Leu Arg Ser Cys Ile Leu Asn 165 170 175 Gly Thr Val Leu Leu AspMet His Ala Asn Thr Ile Glu Glu Ile Ala 180 185 190 Asp Met Val Leu AspGln Gln Val Ser Ser Gly Gln Leu Asn Glu Asp 195 200 205 Val Arg His ArgVal His Glu Ala Leu Met Lys Gln His His His Gln 210 215 220 Asn Gln LysLys Leu Ala Asn Arg Ile Pro Ile Val Arg Ser Leu Ala 225 230 235 240 AspIle Gly Lys Lys Gln Ser Glu Pro Asn Ser Met Asp Lys Asn Ala 245 250 255Gly Gln Val Val Ser Pro Gln Ser Ala Pro Ala Cys Ala Glu Asn Lys 260 265270 Asn Asp Val Ser Arg Glu Asn Ser Thr Val Asp Phe Ser Lys Val Asp 275280 285 Leu His Phe Met Lys Lys Ile Pro Pro Gly Ala Glu Ala Ser Asn Ile290 295 300 Leu Val Gly Glu Leu Glu Phe Leu Asp Arg Ala Val Val Ala PheVal 305 310 315 320 Arg Leu Ser Pro Ala Val Leu Leu Gln Gly Leu Ala GluVal Pro Ile 325 330 335 Pro Ser Arg Phe Leu Phe Ile Leu Leu Gly Pro LeuGly Lys Gly Gln 340 345 350 Gln Tyr His Glu Ile Gly Arg Ser Ile Ala ThrLeu Met Thr Asp Glu 355 360 365 Val Phe His Asp Val Ala Tyr Lys Ala LysAsp Arg Asn Asp Leu Val 370 375 380 Ser Gly Ile Asp Glu Phe Leu Asp GlnVal Thr Val Leu Pro Pro Gly 385 390 395 400 Glu Trp Asp Pro Ser Ile ArgIle Glu Pro Pro Lys Asn Val Pro Ser 405 410 415 Gln Glu Lys Arg Lys IlePro Ala Val Pro Asn Gly Thr Ala Ala His 420 425 430 Gly Glu Ala Glu ProHis Gly Gly His Ser Gly Pro Glu Leu Gln Arg 435 440 445 Thr Gly Arg IlePhe Gly Gly Leu Met Leu Asp Ile Lys Arg Lys Ala 450 455 460 Pro Phe PheTrp Ser Asp Phe Arg Asp Ala Phe Ser Leu Gln Cys Leu 465 470 475 480 AlaSer Phe Leu Phe Leu Tyr Cys Ala Cys Met Ser Pro Val Ile Thr 485 490 495Phe Gly Gly Leu Leu Gly Glu Ala Thr Glu Gly Arg Ile Ser Ala Ile 500 505510 Glu Ser Leu Phe Gly Ala Ser Met Thr Gly Ile Ala Tyr Ser Leu Phe 515520 525 Gly Gly Gln Pro Leu Thr Ile Leu Gly Ser Thr Gly Pro Val Leu Val530 535 540 Phe Glu Lys Ile Leu Phe Lys Phe Cys Lys Glu Tyr Gly Leu SerTyr 545 550 555 560 Leu Ser Leu Arg Ala Ser Ile Gly Leu Trp Thr Ala ThrLeu Cys Ile 565 570 575 Ile Leu Val Ala Thr Asp Ala Ser Ser Leu Val CysTyr Ile Thr Arg 580 585 590 Phe Thr Glu Glu Ala Phe Ala Ser Leu Ile CysIle Ile Phe Ile Tyr 595 600 605 Glu Ala Leu Glu Lys Leu Phe Glu Leu SerGlu Thr Tyr Pro Ile Asn 610 615 620 Met His Asn Asp Leu Glu Leu Leu ThrGln Tyr Ser Cys Asn Cys Met 625 630 635 640 Glu Pro His Ser Pro Ser AsnAsp Thr Leu Lys Glu Trp Arg Glu Ser 645 650 655 Asn Leu Ser Ala Ser AspIle Ile Trp Gly Asn Leu Thr Val Ser Glu 660 665 670 Cys Arg Ser Leu HisGly Glu Tyr Val Gly Arg Ala Cys Gly His Gly 675 680 685 His Pro Tyr ValPro Asp Val Leu Phe Trp Ser Val Ile Leu Phe Phe 690 695 700 Ser Thr ValThr Met Ser Ala Thr Leu Lys Gln Phe Lys Thr Ser Arg 705 710 715 720 TyrPhe Pro Thr Lys Val Arg Ser Ile Val Ser Asp Phe Ala Val Phe 725 730 735Leu Thr Ile Leu Cys Met Val Leu Ile Asp Tyr Ala Ile Gly Ile Pro 740 745750 Ser Pro Lys Leu Gln Val Pro Ser Val Phe Lys Pro Thr Ile Tyr Asp 755760 765 Arg Gly Trp Phe Val Thr Pro Leu Gly Pro Asn Pro Trp Trp Thr Ile770 775 780 Ile Ala Ala Ile Ile Pro Ala Leu Leu Cys Thr Ile Leu Ile PheMet 785 790 795 800 Asp Gln Gln Ile Thr Ala Val Ile Ile Asn Arg Lys GluHis Lys Leu 805 810 815 Lys Lys Gly Cys Gly Tyr His Leu Asp Leu Leu MetVal Ala Val Met 820 825 830 Leu Gly Val Cys Ser Ile Met Gly Leu Pro TrpPhe Val Ala Ala Thr 835 840 845 Val Leu Ser Ile Thr His Val Asn Ser LeuLys Leu Glu Ser Glu Cys 850 855 860 Ser Ala Pro Gly Glu Gln Pro Lys PheLeu Gly Ile Arg Glu Gln Arg 865 870 875 880 Val Thr Gly Leu Met Ile PheIle Leu Met Gly Ser Ser Val Phe Met 885 890 895 Thr Ser Ile Leu Lys PheIle Pro Met Pro Val Leu Tyr Gly Val Phe 900 905 910 Leu Tyr Met Gly AlaSer Ser Leu Lys Gly Ile Gln Leu Phe Asp Arg 915 920 925 Ile Lys Leu PheTrp Met Pro Ala Lys His Gln Pro Asp Phe Ile Tyr 930 935 940 Leu Arg HisVal Pro Leu Arg Lys Val His Leu Phe Thr Val Ile Gln 945 950 955 960 MetSer Cys Leu Gly Leu Leu Trp Ile Ile Lys Val Ser Arg Ala Ala 965 970 975Ile Val Phe Pro Met Met Val Leu Ala Leu Val Phe Val Arg Lys Leu 980 985990 Met Asp Phe Leu Phe Thr Lys Arg Glu Leu Ser Trp Leu Asp Asp Leu 9951000 1005 Met Pro Glu Ser Lys Lys Lys Lys Leu Glu Asp Ala Glu Lys GluGlu 1010 1015 1020 Glu Gln Ser Met Leu Ala Met Glu Asp Glu Gly Thr ValGln Leu Pro 1025 1030 1035 1040 Leu Glu Gly His Tyr Arg Asp Asp Pro SerVal Ile Asn Ile Ser Asp 1045 1050 1055 Glu Met Ser Lys Thr Ala Met TrpGly Asn Leu Leu Val Thr Ala Asp 1060 1065 1070 Asn Ser Lys Glu Lys GluSer Arg Phe Pro Ser Lys Ser Ser Pro Ser 1075 1080 1085 3 4138 DNA Homosapience 3 taagcagagc gagtgccggg ctgagtgtaa gacactgaag acactgcagagcaaggtgct 60 tattccagag gcgttacaaa ac atg gag att aaa gac cag gga gcccaa atg 112 Met Glu Ile Lys Asp Gln Gly Ala Gln Met 1 5 10 gag ccg ctgctg cct acg aga aat gat gaa gaa gca gtt gtg gat aga 160 Glu Pro Leu LeuPro Thr Arg Asn Asp Glu Glu Ala Val Val Asp Arg 15 20 25 ggt gga act cgttct att ctc aaa aca cac ttt gag aaa gaa gat tta 208 Gly Gly Thr Arg SerIle Leu Lys Thr His Phe Glu Lys Glu Asp Leu 30 35 40 gaa ggt cat cga acacta ttt att gga gta cat gtg ccc ttg gga gga 256 Glu Gly His Arg Thr LeuPhe Ile Gly Val His Val Pro Leu Gly Gly 45 50 55 aga aaa agc cat cga cgtcac agg cat cgt ggt cat aaa cac aga aag 304 Arg Lys Ser His Arg Arg HisArg His Arg Gly His Lys His Arg Lys 60 65 70 aga gac aga gaa aga gat tcagga tta gag gat gga agg gag tca cct 352 Arg Asp Arg Glu Arg Asp Ser GlyLeu Glu Asp Gly Arg Glu Ser Pro 75 80 85 90 tct ttt gac acc cca tca cagagg gta cag ttt att ctt gga acc gag 400 Ser Phe Asp Thr Pro Ser Gln ArgVal Gln Phe Ile Leu Gly Thr Glu 95 100 105 gat gat gac gag gaa cac attcct cat gac ctt ttc aca gaa ctg gat 448 Asp Asp Asp Glu Glu His Ile ProHis Asp Leu Phe Thr Glu Leu Asp 110 115 120 gag att tgt tgg cgt gaa ggtgag gac gct gag tgg cga gaa aca gcc 496 Glu Ile Cys Trp Arg Glu Gly GluAsp Ala Glu Trp Arg Glu Thr Ala 125 130 135 agg tgg ttg aag ttt gaa gaagat gtg gaa gat gga gga gaa agg tgg 544 Arg Trp Leu Lys Phe Glu Glu AspVal Glu Asp Gly Gly Glu Arg Trp 140 145 150 agc aag cct tat gtg gct actctt tca ttg cac agc ttg ttt gaa ttg 592 Ser Lys Pro Tyr Val Ala Thr LeuSer Leu His Ser Leu Phe Glu Leu 155 160 165 170 aga agt tgt att ctg aatgga act gtg ttg ctg gac atg cat gcc aac 640 Arg Ser Cys Ile Leu Asn GlyThr Val Leu Leu Asp Met His Ala Asn 175 180 185 act tta gaa gaa att gcagat atg gtt ctt gac caa caa gtg agc tca 688 Thr Leu Glu Glu Ile Ala AspMet Val Leu Asp Gln Gln Val Ser Ser 190 195 200 ggt cag ctg aat gaa gatgta cgc cat agg gtc cat gag gca ttg atg 736 Gly Gln Leu Asn Glu Asp ValArg His Arg Val His Glu Ala Leu Met 205 210 215 aaa cag cat cat cat cagaat cag aaa aaa ctc acc aac agg att ccc 784 Lys Gln His His His Gln AsnGln Lys Lys Leu Thr Asn Arg Ile Pro 220 225 230 att gtt cgt tcc ttt gctgat att ggc aag aaa cag tca gaa cca aat 832 Ile Val Arg Ser Phe Ala AspIle Gly Lys Lys Gln Ser Glu Pro Asn 235 240 245 250 tcc atg gac aaa aatgca ggt cag gtt gtt tct cct cag tct gct cca 880 Ser Met Asp Lys Asn AlaGly Gln Val Val Ser Pro Gln Ser Ala Pro 255 260 265 gcc tgt gtt gaa aataaa aat gat gtt agc aga gaa aac agc act gtt 928 Ala Cys Val Glu Asn LysAsn Asp Val Ser Arg Glu Asn Ser Thr Val 270 275 280 gac ttt agc aag gttgat ctg cat ttt atg aaa aag att cct cca ggt 976 Asp Phe Ser Lys Val AspLeu His Phe Met Lys Lys Ile Pro Pro Gly 285 290 295 gct gaa gca tcg aacatc tta ctg gga gaa ctg gag ttc ttg gat cga 1024 Ala Glu Ala Ser Asn IleLeu Leu Gly Glu Leu Glu Phe Leu Asp Arg 300 305 310 aca gta gtt gcg tttgtc agg ttg tct cca gct gta ttg ctt caa gga 1072 Thr Val Val Ala Phe ValArg Leu Ser Pro Ala Val Leu Leu Gln Gly 315 320 325 330 ctg gct gaa gtccca atc cca acc aga ttt ttg ttc att ctt ctg gga 1120 Leu Ala Glu Val ProIle Pro Thr Arg Phe Leu Phe Ile Leu Leu Gly 335 340 345 ccc ctg gga aagggt caa cag tac cat gag att ggc aga tca att gca 1168 Pro Leu Gly Lys GlyGln Gln Tyr His Glu Ile Gly Arg Ser Ile Ala 350 355 360 acc cta atg acagat gag gta ttt cat gat gtt gcc tat aaa gct aaa 1216 Thr Leu Met Thr AspGlu Val Phe His Asp Val Ala Tyr Lys Ala Lys 365 370 375 gat cgt aat gacttg gta tca gga att gat gag ttt ctg gat cag gtt 1264 Asp Arg Asn Asp LeuVal Ser Gly Ile Asp Glu Phe Leu Asp Gln Val 380 385 390 act gtt ctc cctcct gga gaa tgg gat cca agc att cga ata gag cct 1312 Thr Val Leu Pro ProGly Glu Trp Asp Pro Ser Ile Arg Ile Glu Pro 395 400 405 410 ccc aaa aatgtt cct tcc cag gag aag agg aag att cct gct gta cca 1360 Pro Lys Asn ValPro Ser Gln Glu Lys Arg Lys Ile Pro Ala Val Pro 415 420 425 aat gga acagca gct cat ggg gaa gca gag ccc cac gga gga cat agt 1408 Asn Gly Thr AlaAla His Gly Glu Ala Glu Pro His Gly Gly His Ser 430 435 440 gga cct gaactc cag cga act gga agg att ttt ggg gga ctt att tta 1456 Gly Pro Glu LeuGln Arg Thr Gly Arg Ile Phe Gly Gly Leu Ile Leu 445 450 455 gat atc aaaaga aaa gct cca tac ttc tgg agt gac ttc aga gat gct 1504 Asp Ile Lys ArgLys Ala Pro Tyr Phe Trp Ser Asp Phe Arg Asp Ala 460 465 470 ttc agc ctgcag tgc tta gca tct ttt cta ttt ctc tac tgc gcg tgt 1552 Phe Ser Leu GlnCys Leu Ala Ser Phe Leu Phe Leu Tyr Cys Ala Cys 475 480 485 490 atg tctcct gtc atc acg ttt gga gga ctg ctg gga gaa gca act gaa 1600 Met Ser ProVal Ile Thr Phe Gly Gly Leu Leu Gly Glu Ala Thr Glu 495 500 505 ggg cgtata agt gca att gaa tct ctc ttt gga gca tcc atg acc ggg 1648 Gly Arg IleSer Ala Ile Glu Ser Leu Phe Gly Ala Ser Met Thr Gly 510 515 520 ata gcctat tct ctc ttt ggt gga cag cct ctt acc ata tta ggc agt 1696 Ile Ala TyrSer Leu Phe Gly Gly Gln Pro Leu Thr Ile Leu Gly Ser 525 530 535 aca ggacca gtt ttg gtg ttt gaa aag att ttg ttt aaa ttt tgc aaa 1744 Thr Gly ProVal Leu Val Phe Glu Lys Ile Leu Phe Lys Phe Cys Lys 540 545 550 gaa tatggg ctg tca tac cta tct tta aga gct agc att gga ctt tgg 1792 Glu Tyr GlyLeu Ser Tyr Leu Ser Leu Arg Ala Ser Ile Gly Leu Trp 555 560 565 570 actgca act cta tgt atc ata ctt gtg gcc aca gat gct agt tcc ctt 1840 Thr AlaThr Leu Cys Ile Ile Leu Val Ala Thr Asp Ala Ser Ser Leu 575 580 585 gtctgc tac atc act cgg ttt act gaa gaa gct ttt gct tcc ctg att 1888 Val CysTyr Ile Thr Arg Phe Thr Glu Glu Ala Phe Ala Ser Leu Ile 590 595 600 tgcatc att ttc att tat gag gcc ctg gag aag ttg ttt gaa ctc agt 1936 Cys IleIle Phe Ile Tyr Glu Ala Leu Glu Lys Leu Phe Glu Leu Ser 605 610 615 gaagca tat cca atc aac atg cat aat gat ctg gaa ctg ctg aca caa 1984 Glu AlaTyr Pro Ile Asn Met His Asn Asp Leu Glu Leu Leu Thr Gln 620 625 630 tactcg tgt aac tgt gtg gaa ccg cat aat ccc agc aat ggc aca ttg 2032 Tyr SerCys Asn Cys Val Glu Pro His Asn Pro Ser Asn Gly Thr Leu 635 640 645 650aag gaa tgg agg gaa tcc aat att tct gcc tct gac ata att tgg gag 2080 LysGlu Trp Arg Glu Ser Asn Ile Ser Ala Ser Asp Ile Ile Trp Glu 655 660 665aac cta act gtg tca gaa tgc aaa tca ttg cat gga gag tat gtt gga 2128 AsnLeu Thr Val Ser Glu Cys Lys Ser Leu His Gly Glu Tyr Val Gly 670 675 680cgg gcc tgt ggc cat gat cac cca tat gtt cca gat gtt cta ttt tgg 2176 ArgAla Cys Gly His Asp His Pro Tyr Val Pro Asp Val Leu Phe Trp 685 690 695tct gtg atc ctg ttc ttt tcc aca gtt act ctg tca gcc acc ctg aag 2224 SerVal Ile Leu Phe Phe Ser Thr Val Thr Leu Ser Ala Thr Leu Lys 700 705 710cag ttc aag act agc aga tat ttt cca acc aag gtt cga tcc ata gtg 2272 GlnPhe Lys Thr Ser Arg Tyr Phe Pro Thr Lys Val Arg Ser Ile Val 715 720 725730 agt gac ttt gct gtc ttt ctt aca att ctg tgt atg gtt tta att gac 2320Ser Asp Phe Ala Val Phe Leu Thr Ile Leu Cys Met Val Leu Ile Asp 735 740745 tat gcc att ggg atc cca tct cca aaa cta caa gta cca agt gtt ttc 2368Tyr Ala Ile Gly Ile Pro Ser Pro Lys Leu Gln Val Pro Ser Val Phe 750 755760 aag ccc act aga gat gat cgt ggc tgg ttt gtt acg cct tta ggt cca 2416Lys Pro Thr Arg Asp Asp Arg Gly Trp Phe Val Thr Pro Leu Gly Pro 765 770775 aac cca tgg tgg aca gta ata gct gct ata att cca gct ctg ctt tgt 2464Asn Pro Trp Trp Thr Val Ile Ala Ala Ile Ile Pro Ala Leu Leu Cys 780 785790 act att cta att ttc atg gac caa cag att aca gct gtc atc atc aac 2512Thr Ile Leu Ile Phe Met Asp Gln Gln Ile Thr Ala Val Ile Ile Asn 795 800805 810 agg aaa gag cat aag cta aag aaa ggt tgt ggg tac cat ctg gac cta2560 Arg Lys Glu His Lys Leu Lys Lys Gly Cys Gly Tyr His Leu Asp Leu 815820 825 tta atg gtg gct gtc atg ctc ggt gta tgc tcc atc atg ggc ctg cca2608 Leu Met Val Ala Val Met Leu Gly Val Cys Ser Ile Met Gly Leu Pro 830835 840 tgg ttt gtg gct gcc aca gtc ctc tcc atc act cat gtc aat agc cta2656 Trp Phe Val Ala Ala Thr Val Leu Ser Ile Thr His Val Asn Ser Leu 845850 855 aaa ctg gaa tca gaa tgc tca gct cca gga gaa caa ccc aaa ttt ctc2704 Lys Leu Glu Ser Glu Cys Ser Ala Pro Gly Glu Gln Pro Lys Phe Leu 860865 870 ggc att cgg gag caa agg gtt act ggg ctt atg att ttt att ctt atg2752 Gly Ile Arg Glu Gln Arg Val Thr Gly Leu Met Ile Phe Ile Leu Met 875880 885 890 ggt tca tca gtc ttt atg acc agt att ctg aag ttt att ccc atgcca 2800 Gly Ser Ser Val Phe Met Thr Ser Ile Leu Lys Phe Ile Pro Met Pro895 900 905 gtg cta tat gga gtg ttt ctt tat atg ggt gct tca tct cta aaggga 2848 Val Leu Tyr Gly Val Phe Leu Tyr Met Gly Ala Ser Ser Leu Lys Gly910 915 920 att cag ttc ttt gat agg ata aag ctc ttc tgg atg ccg gca aaacat 2896 Ile Gln Phe Phe Asp Arg Ile Lys Leu Phe Trp Met Pro Ala Lys His925 930 935 caa cca gat ttt ata tac cta agg cac gta ccg ctt cga aaa gtgcat 2944 Gln Pro Asp Phe Ile Tyr Leu Arg His Val Pro Leu Arg Lys Val His940 945 950 ctc ttc aca att att cag atg agt tgc ctt ggc ctt ttg tgg ataata 2992 Leu Phe Thr Ile Ile Gln Met Ser Cys Leu Gly Leu Leu Trp Ile Ile955 960 965 970 aaa gtt tca aga gct gct att gtc tct ccc atg atg gtg ttatcc ctg 3040 Lys Val Ser Arg Ala Ala Ile Val Ser Pro Met Met Val Leu SerLeu 975 980 985 gtt ttt gta aga aag ttg atg gac ttg ttg ttc acg aaa cgggaa ctc 3088 Val Phe Val Arg Lys Leu Met Asp Leu Leu Phe Thr Lys Arg GluLeu 990 995 1000 tgc tgg ttg gat gat ttg atg cct gag agt aag aaa aag aaactg gaa 3136 Cys Trp Leu Asp Asp Leu Met Pro Glu Ser Lys Lys Lys Lys LeuGlu 1005 1010 1015 tat gct gaa aaa gaa gaa gaa caa tgt gtg cta cct atggaa gat gag 3184 Tyr Ala Glu Lys Glu Glu Glu Gln Cys Val Leu Pro Met GluAsp Glu 1020 1025 1030 ggc aca gta caa ctc cca ttg gaa ggg cac tat agagat gat cca tct 3232 Gly Thr Val Gln Leu Pro Leu Glu Gly His Tyr Arg AspAsp Pro Ser 1035 1040 1045 1050 gtg atc aat ata tct gat gaa atg tca aagact gcc ttg tgg agg aac 3280 Val Ile Asn Ile Ser Asp Glu Met Ser Lys ThrAla Leu Trp Arg Asn 1055 1060 1065 ctt ctg att act gcc gat aac tca aaagat aag gag tca agc ttt cct 3328 Leu Leu Ile Thr Ala Asp Asn Ser Lys AspLys Glu Ser Ser Phe Pro 1070 1075 1080 tcc aaa agc tcc cct tcc taatcactctaga agctgattcc ccaaagcatt 3379 Ser Lys Ser Ser Pro Ser 1085gaaagccgaa aagagaagaa agctgactca gggatagttg ttgacaggga gacttgtcta 3439tgactcgatc ttcaatttat tttttacata tatatgagaa gagtgtcaca attattaata 3499aaactgcttg gatcatgtat ggtaaattct gtccctcaac ccaaatccac tttcatacgg 3559taagtagggc aaaacttgtt tcatttcggt gttaaaattt cggagcagga gacatccctg 3619tgagcagaaa caatagccaa tgcagaatct gtgtgttcct tgctgaacgt aagacatttg 3679taaactggat tctgattgtc agttttatga gagcaatagc ttccttaaag agataagtca 3739tatacaccta gtttgtattc tcatacttta gagacctgaa gacgcctgat aatttcattc 3799aggagaattt ttgaaaggta gtcaaacttc tttttagttt ttatagctta gcattagtga 3859cttatttcaa aagacccaaa tcaaaaagtt agtttgaaag cattttttaa taattgtatt 3919tatgcatttg gctactgtaa gttttgctcc atggaataat gatgtgatag caaaaatgaa 3979taagactatg aataagttcc tacatgaagg ttaatgtcag tggtgaaaaa tcttattatg 4039ctccaatata ctgccagcat gctgagtata cttggatcat aaaaaactgt ttcatttttc 4099ttatttattt tatgcatagg aatattcatt ccggaattc 4138 4 1088 PRT Homo sapience4 Met Glu Ile Lys Asp Gln Gly Ala Gln Met Glu Pro Leu Leu Pro Thr 1 5 1015 Arg Asn Asp Glu Glu Ala Val Val Asp Arg Gly Gly Thr Arg Ser Ile 20 2530 Leu Lys Thr His Phe Glu Lys Glu Asp Leu Glu Gly His Arg Thr Leu 35 4045 Phe Ile Gly Val His Val Pro Leu Gly Gly Arg Lys Ser His Arg Arg 50 5560 His Arg His Arg Gly His Lys His Arg Lys Arg Asp Arg Glu Arg Asp 65 7075 80 Ser Gly Leu Glu Asp Gly Arg Glu Ser Pro Ser Phe Asp Thr Pro Ser 8590 95 Gln Arg Val Gln Phe Ile Leu Gly Thr Glu Asp Asp Asp Glu Glu His100 105 110 Ile Pro His Asp Leu Phe Thr Glu Leu Asp Glu Ile Cys Trp ArgGlu 115 120 125 Gly Glu Asp Ala Glu Trp Arg Glu Thr Ala Arg Trp Leu LysPhe Glu 130 135 140 Glu Asp Val Glu Asp Gly Gly Glu Arg Trp Ser Lys ProTyr Val Ala 145 150 155 160 Thr Leu Ser Leu His Ser Leu Phe Glu Leu ArgSer Cys Ile Leu Asn 165 170 175 Gly Thr Val Leu Leu Asp Met His Ala AsnThr Leu Glu Glu Ile Ala 180 185 190 Asp Met Val Leu Asp Gln Gln Val SerSer Gly Gln Leu Asn Glu Asp 195 200 205 Val Arg His Arg Val His Glu AlaLeu Met Lys Gln His His His Gln 210 215 220 Asn Gln Lys Lys Leu Thr AsnArg Ile Pro Ile Val Arg Ser Phe Ala 225 230 235 240 Asp Ile Gly Lys LysGln Ser Glu Pro Asn Ser Met Asp Lys Asn Ala 245 250 255 Gly Gln Val ValSer Pro Gln Ser Ala Pro Ala Cys Val Glu Asn Lys 260 265 270 Asn Asp ValSer Arg Glu Asn Ser Thr Val Asp Phe Ser Lys Val Asp 275 280 285 Leu HisPhe Met Lys Lys Ile Pro Pro Gly Ala Glu Ala Ser Asn Ile 290 295 300 LeuLeu Gly Glu Leu Glu Phe Leu Asp Arg Thr Val Val Ala Phe Val 305 310 315320 Arg Leu Ser Pro Ala Val Leu Leu Gln Gly Leu Ala Glu Val Pro Ile 325330 335 Pro Thr Arg Phe Leu Phe Ile Leu Leu Gly Pro Leu Gly Lys Gly Gln340 345 350 Gln Tyr His Glu Ile Gly Arg Ser Ile Ala Thr Leu Met Thr AspGlu 355 360 365 Val Phe His Asp Val Ala Tyr Lys Ala Lys Asp Arg Asn AspLeu Val 370 375 380 Ser Gly Ile Asp Glu Phe Leu Asp Gln Val Thr Val LeuPro Pro Gly 385 390 395 400 Glu Trp Asp Pro Ser Ile Arg Ile Glu Pro ProLys Asn Val Pro Ser 405 410 415 Gln Glu Lys Arg Lys Ile Pro Ala Val ProAsn Gly Thr Ala Ala His 420 425 430 Gly Glu Ala Glu Pro His Gly Gly HisSer Gly Pro Glu Leu Gln Arg 435 440 445 Thr Gly Arg Ile Phe Gly Gly LeuIle Leu Asp Ile Lys Arg Lys Ala 450 455 460 Pro Tyr Phe Trp Ser Asp PheArg Asp Ala Phe Ser Leu Gln Cys Leu 465 470 475 480 Ala Ser Phe Leu PheLeu Tyr Cys Ala Cys Met Ser Pro Val Ile Thr 485 490 495 Phe Gly Gly LeuLeu Gly Glu Ala Thr Glu Gly Arg Ile Ser Ala Ile 500 505 510 Glu Ser LeuPhe Gly Ala Ser Met Thr Gly Ile Ala Tyr Ser Leu Phe 515 520 525 Gly GlyGln Pro Leu Thr Ile Leu Gly Ser Thr Gly Pro Val Leu Val 530 535 540 PheGlu Lys Ile Leu Phe Lys Phe Cys Lys Glu Tyr Gly Leu Ser Tyr 545 550 555560 Leu Ser Leu Arg Ala Ser Ile Gly Leu Trp Thr Ala Thr Leu Cys Ile 565570 575 Ile Leu Val Ala Thr Asp Ala Ser Ser Leu Val Cys Tyr Ile Thr Arg580 585 590 Phe Thr Glu Glu Ala Phe Ala Ser Leu Ile Cys Ile Ile Phe IleTyr 595 600 605 Glu Ala Leu Glu Lys Leu Phe Glu Leu Ser Glu Ala Tyr ProIle Asn 610 615 620 Met His Asn Asp Leu Glu Leu Leu Thr Gln Tyr Ser CysAsn Cys Val 625 630 635 640 Glu Pro His Asn Pro Ser Asn Gly Thr Leu LysGlu Trp Arg Glu Ser 645 650 655 Asn Ile Ser Ala Ser Asp Ile Ile Trp GluAsn Leu Thr Val Ser Glu 660 665 670 Cys Lys Ser Leu His Gly Glu Tyr ValGly Arg Ala Cys Gly His Asp 675 680 685 His Pro Tyr Val Pro Asp Val LeuPhe Trp Ser Val Ile Leu Phe Phe 690 695 700 Ser Thr Val Thr Leu Ser AlaThr Leu Lys Gln Phe Lys Thr Ser Arg 705 710 715 720 Tyr Phe Pro Thr LysVal Arg Ser Ile Val Ser Asp Phe Ala Val Phe 725 730 735 Leu Thr Ile LeuCys Met Val Leu Ile Asp Tyr Ala Ile Gly Ile Pro 740 745 750 Ser Pro LysLeu Gln Val Pro Ser Val Phe Lys Pro Thr Arg Asp Asp 755 760 765 Arg GlyTrp Phe Val Thr Pro Leu Gly Pro Asn Pro Trp Trp Thr Val 770 775 780 IleAla Ala Ile Ile Pro Ala Leu Leu Cys Thr Ile Leu Ile Phe Met 785 790 795800 Asp Gln Gln Ile Thr Ala Val Ile Ile Asn Arg Lys Glu His Lys Leu 805810 815 Lys Lys Gly Cys Gly Tyr His Leu Asp Leu Leu Met Val Ala Val Met820 825 830 Leu Gly Val Cys Ser Ile Met Gly Leu Pro Trp Phe Val Ala AlaThr 835 840 845 Val Leu Ser Ile Thr His Val Asn Ser Leu Lys Leu Glu SerGlu Cys 850 855 860 Ser Ala Pro Gly Glu Gln Pro Lys Phe Leu Gly Ile ArgGlu Gln Arg 865 870 875 880 Val Thr Gly Leu Met Ile Phe Ile Leu Met GlySer Ser Val Phe Met 885 890 895 Thr Ser Ile Leu Lys Phe Ile Pro Met ProVal Leu Tyr Gly Val Phe 900 905 910 Leu Tyr Met Gly Ala Ser Ser Leu LysGly Ile Gln Phe Phe Asp Arg 915 920 925 Ile Lys Leu Phe Trp Met Pro AlaLys His Gln Pro Asp Phe Ile Tyr 930 935 940 Leu Arg His Val Pro Leu ArgLys Val His Leu Phe Thr Ile Ile Gln 945 950 955 960 Met Ser Cys Leu GlyLeu Leu Trp Ile Ile Lys Val Ser Arg Ala Ala 965 970 975 Ile Val Ser ProMet Met Val Leu Ser Leu Val Phe Val Arg Lys Leu 980 985 990 Met Asp LeuLeu Phe Thr Lys Arg Glu Leu Cys Trp Leu Asp Asp Leu 995 1000 1005 MetPro Glu Ser Lys Lys Lys Lys Leu Glu Tyr Ala Glu Lys Glu Glu 1010 10151020 Glu Gln Cys Val Leu Pro Met Glu Asp Glu Gly Thr Val Gln Leu Pro1025 1030 1035 1040 Leu Glu Gly His Tyr Arg Asp Asp Pro Ser Val Ile AsnIle Ser Asp 1045 1050 1055 Glu Met Ser Lys Thr Ala Leu Trp Arg Asn LeuLeu Ile Thr Ala Asp 1060 1065 1070 Asn Ser Lys Asp Lys Glu Ser Ser PhePro Ser Lys Ser Ser Pro Ser 1075 1080 1085 5 19 DNA Homo sapience 5tttggagaaa acccctggt 19 6 20 DNA Homo sapience 6 tgacatcatc caggaagctg20 7 20 DNA Homo sapience 7 gtcatgttag accaacaggt 20 8 18 DNA Homosapience 8 gttgtaatag cgacactc 18

What is claimed is:
 1. A protein comprising the amino acid sequence setforth as SEQ ID NO: 2 or NO: 4 in the Sequence Listing.
 2. A proteincomprising an amino acid sequence having deletion, substitution,addition or insertion of one or more amino acids relative to the aminoacid sequence set forth as SEQ ID NO: 2 or NO: 4 in the SequenceListing, which protein, when expressed in a cell, functions as sodiumion-driven chloride/bicarbonate exchanger.
 3. The protein of claim 2wherein the sodium ion-driven chloride/bicarbonate exchanger,dependently upon both of extracellular bicarbonate and intracellularchloride ions, takes up extracellular sodium ion into the cell andtransport intracellular sodium ion out of the cell.
 4. A cell in whichone of the proteins of claims 1 to 3 is expressed, wherein the cell isof a species different from the species of origin of the one of theproteins.
 5. An antibody to one of the proteins of claims 1 to
 3. 6. Amethod for selection of agonists and antagonists of sodium ion-drivenchloride/bicarbonate exchanger, which method comprises bringing the cellof claim 4 into contact with a candidate compound, measuring thefunction of the sodium ion-driven chloride/bicarbonate exchanger,comparing the result thus obtained with a result obtained by measuringthe function of the sodium ion-driven chloride/bicarbonate exchanger ofthe cell of claim 4 which has not been brought into contact with thecandidate compound, and thereby determining whether or not the candidatecompound enhances or inhibits the function.
 7. A DNA comprising thenucleotide sequence set forth as SEQ ID NO: 1 or NO: 3 in the SequenceListing.
 8. A DNA comprising a nucleotide sequence consisting of thenucleotides 67 through 3330 in the nucleotide sequence set forth as SEQID NO: 1 in the Sequence Listing.
 9. A DNA comprising a nucleotidesequence consisting of the nucleotides 83 through 3346 in the nucleotidesequence set forth as SEQ ID NO: 3 in the Sequence Listing.
 10. A DNAcomprising a nucleotide sequence having deletion, substitution, additionor insertion of one or more nucleotides relative to a DNA comprising anucleotide sequence consisting of the nucleotides 67 through 3330 in thenucleotide sequence set forth as SEQ ID NO: 1 in the Sequence Listing,and encoding: (1) a protein comprising the amino acid sequence set forthas SEQ ID NO: 2 in the Sequence Listing, or (2) a protein comprising anamino acid sequence having deletion, substitution, addition or insertionof one or more amino acids relative to the amino acid sequence set forthas SEQ ID NO: 2 in the Sequence Listing, which protein, when expressedin a cell, functions as sodium ion-driven chloride/bicarbonateexchanger.
 11. A DNA comprising a nucleotide sequence having deletion,substitution, addition or insertion of one or more nucleotides relativeto a DNA comprising a nucleotide sequence consisting of the nucleotides83 through 3346 in the nucleotide sequence set forth as SEQ ID NO: 3 inthe Sequence Listing, and encoding: (1) a protein comprising the aminoacid sequence set forth as SEQ ID NO: 4 in the Sequence Listing, or (2)a protein comprising an amino acid sequence having deletion,substitution, addition or insertion of one or more amino acids relativeto the amino acid sequence set forth as SEQ ID NO: 4 in the SequenceListing, which protein, when expressed in a cell, functions as sodiumion-driven chloride/bicarbonate exchanger.